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Biomedical subjects

J Lukas

Publications and source records attributed to J Lukas.

At least 37 records · Page 2Linked to original sources
PubMed2001

Inhibitory effects of 1alpha,25-dihydroxyvitamin D(3) on the G(1)-S phase-controlling machinery.

The nuclear hormone 1alpha,25-dihydroxyvitamin D(3) induces cell cycle arrest, differentiation, or apoptosis depending on target cell type and state. Although the antiproliferative effect of 1alpha,25-dihydroxyvitamin D(3) has been known for years, the molecular basis of the cell cycle blockade by 1alpha,25-dihydroxyvitamin D(3) remains largely unknown. Here we have investigated the mechanisms underlying the G(1) arrest induced upon 1alpha,25-dihydroxyvitamin D(3) treatment of the human breast cancer cell line MCF-7. Twenty-four-hour exposure of exponentially growing MCF-7 cells to 1alpha,25-dihydroxyvitamin D(3) impeded proliferation by preventing S phase entry, an effect that correlated with appearance of the growth-suppressing, hypophosphorylated form of the retinoblastoma protein (pRb), and modulation of cyclin-dependent kinase (cdk) activities of cdk-4, -6, and -2. Time course immunochemical and biochemical analyses of the cellular and molecular effects of 1alpha,25-dihydroxyvitamin D(3) treatment for up to 6 d revealed a dynamic chain of events, preventing activation of cyclin D1/cdk4, and loss of cyclin D3, which collectively lead to repression of the E2F transcription factors and thus negatively affected cyclin A protein expression. While the observed 10-fold inhibition of cyclin D1/cdk 4-associated kinase activity appeared independent of cdk inhibitors, the activity of cdk 2 decreased about 20-fold, reflecting joint effects of the lower abundance of its cyclin partners and a significant increase of the cdk inhibitor p21(CIP1/WAF1), which blocked the remaining cyclin A(E)/cdk 2 complexes. Together with a rapid down-modulation of the c-Myc oncoprotein in response to 1alpha,25-dihydroxyvitamin D(3), these results demonstrate that 1alpha,25-dihydroxyvitamin D(3) inhibits cell proliferation by targeting several key regulators governing the G(1)/S transition.

Breast Neoplasms↗
PubMed2001

16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting.

PURPOSE: Establishment of a new molecular biology technique for the identification of multiple bacteria from the ocular environment, which can be applied supplementarily to cultivation in cases of severe bacterial infections. METHODS: From 60 human conjunctivae (29 with purulent and 31 with nonpurulent conjunctivitis), swabs were taken and DNA was extracted. Fragments of 200 bp, spanning the V3 region of the eubacterial 16S rDNA, were amplified by polymerase chain reaction (PCR) and separated by denaturing gradient gel electrophoresis (DGGE). For phylogenetic identification, DGGE bands were excised and directly sequenced, or 16S rDNA clone libraries were constructed and clones were screened by DGGE. Sequences were compared with sequences of known bacteria listed in the EMBL database. Furthermore, the results were compared with results obtained from conventional cultivation. RESULTS: 16S rDNA could be amplified from 25 of 29 investigated swabs taken from purulent conjunctivitis eyes and from 2 of 31 investigated swabs taken from nonpurulent conjunctivitis eyes. Sixteen samples showed monomicrobial and 11 samples showed polymicrobial infections. The following genera (n is number of samples) were detected: Staphylococcus (n = 8), Corynebacterium (n = 7), Propionibacterium (n = 7), Streptococcus (n = 6), Bacillus (n = 2), Acinetobacter (n = 3), Pseudomonas (n = 3), Proteus (n = 1), and Brevundimonas (n = 1). Four sequences could not be identified to the genus level. They had highest sequence similarities both to sequences of Pantoea and Enterobacter (n = 1), Kingella and Neisseria (n = 1), Serratia and Aranicola (n = 1), and Leuconostoc and Weissella (n = 2), respectively. Culture was only positive for coagulase-negative staphylococci (n = 9), Corynebacteria (n = 3), Staphylococcus aureus (n = 1), Streptococcus sp. (n = 1), Proteus sp. (n = 1), Klebsiella oxytoca (n = 1), and Pseudomonas aeruginosa (n = 1). In total, 45% of the 60 analyzed conjunctival swabs were PCR positive, whereas only 22% were culture positive. No sample positive by culture gave negative results by PCR. CONCLUSIONS: 16S rDNA sequence analyses and DGGE fingerprinting are appropriate methods for the detection and identification of monomicrobial as well as polymicrobial ocular infections of bacteria that might not be detected by conventional cultivation.

Bacteria↗
PubMed2000

Ubiquitin/proteasome-mediated degradation of p19INK4d determines its periodic expression during the cell cycle.

Assembly and activity of the proto-oncogenic cyclin D/CDK4(6) complexes, the major driving force of G1 phase progression, is negatively regulated by a family of INK4 CDK inhibitors p16INK4a, p15INK4b, p18INK4c, and p19INK4d. Expression of the INK4 family members is controlled at the transcriptional level, through differential response to environmental and intracellular signals such as cytokines, oncogenic overload, or cellular senescence. Here we show that the periodic oscillation of the p19INK4d protein during the cell cycle is determined by the ubiquitin/proteasome-dependent mechanism, allowing the protein abundance to follow the changes in its mRNA expression. Within the INK4 family, this regulatory mode appears restricted to p19INK4d whose ubiquitination was dependent on the integrity of lysine 62, and binding to CDK4. These results highlight unexpected differences among the INK4 inhibitors, and suggest how p19INK4d may help regulate the rate of cyclin D/CDK4(6) complex formation, and thereby timely progression through the mammalian cell division cycle. Oncogene (2000) 19, 2870 - 2876

Carrier Proteins↗
PubMed2000

Rapid destruction of human Cdc25A in response to DNA damage.

To protect genome integrity and ensure survival, eukaryotic cells exposed to genotoxic stress cease proliferating to provide time for DNA repair. Human cells responded to ultraviolet light or ionizing radiation by rapid, ubiquitin- and proteasome-dependent protein degradation of Cdc25A, a phosphatase that is required for progression from G1 to S phase of the cell cycle. This response involved activated Chk1 protein kinase but not the p53 pathway, and the persisting inhibitory tyrosine phosphorylation of Cdk2 blocked entry into S phase and DNA replication. Overexpression of Cdc25A bypassed this mechanism, leading to enhanced DNA damage and decreased cell survival. These results identify specific degradation of Cdc25A as part of the DNA damage checkpoint mechanism and suggest how Cdc25A overexpression in human cancers might contribute to tumorigenesis.

CDC2-CDC28 Kinases↗
PubMed2000

Cell cycle arrest by the PTEN tumor suppressor is target cell specific and may require protein phosphatase activity.

PTEN, a tumor suppressor commonly targeted in human cancer, possesses phosphatase activities toward both protein and lipid substrates. While PTEN suppresses gliomas through cell cycle inhibition which requires its lipid phosphatase activity, PTEN's effects on other tumor types and the role of its protein phosphatase activity are controversial or unknown. Here we show that exogenous wild-type PTEN arrests some, but not all human breast cancer cell lines in G1, in a manner independent of endogenous PTEN. Unexpectedly, the G129E mutant of PTEN selectively deficient in the lipid phosphatase activity still blocked the cell cycle of MCF-7 cells, while the G129R and H123Y mutants lacking both phosphatase activities were ineffective. These results suggest that PTEN's protein phosphatase activity likely contributes to its tumor suppressor function in subsets of tumors and that elucidation of downstream targets which dictate cellular responses to PTEN may have important implications for future cancer treatment strategies.

Blotting, Western↗
PubMed2000

Distinct versus redundant properties among members of the INK4 family of cyclin-dependent kinase inhibitors.

p16(INK4a), p15(INK4b), p18(INK4c) and p19(INK4d) comprise a family of cyclin-dependent kinase inhibitors and tumor suppressors. We report that the INK4 proteins share the ability to arrest cells in G1, and interact with CDK4 or CDK6 with similar avidity. In contrast, only p18 and particularly p19 are phosphorylated in vivo, and each of the human INK4 proteins shows unique expression patterns dependent on cell and tissue type, and differentiation stage. Thus, the INK4 proteins harbor redundant as well as non-overlapping properties, suggesting distinct regulatory modes, and diverse roles for the individual INK4 family members in cell cycle control, cellular differentiation, and multistep oncogenesis.

Binding, Competitive↗
PubMed2000

p53 mutations and expression in breast carcinoma in situ.

The p53 tumor suppressor gene is altered in approximately half of human cancers. Although p53 mutations are common in invasive breast carcinoma, few have been identified in breast carcinoma in situ (intraductal breast carcinomas). Most studies of p53 in breast carcinoma in situ are immunohistochemical studies of p53 staining in paraffin-embedded tissue sections. Few studies have isolated the tumor cells and subjected them to DNA sequence analysis. The current study was undertaken to characterize p53 in a cohort of breast carcinoma in situ cases, both with and without invasive disease. Fifty-eight frozen breast biopsy samples were used for these investigations. Twenty-seven cases had only ductal carcinoma in situ (CIS) and 31 cases had evidence of both invasive and in situ carcinoma. DNA sequence alterations in exons 2 through 11 of p53 were screened by the single-strand conformational polymorphism technique. Exons with altered mobility were sequenced. Among breast CIS cases without invasive disease, 22% had p53 mutations and 7% had DNA sequence alterations of unknown significance. Analysis of breast CIS with concurrent invasive disease demonstrated p53 mutations in 19% of cases and one (3%) DNA alteration of unknown significance. Each carcinoma having a p53 mutation in the breast CIS component had the identical mutation in the invasive component of the same tumor indicating a clonal relationship between the two tumor components. p53 protein overexpression was identified in 22% of pure intraductal breast carcinomas and in 35% of breast CIS with invasive disease. Comparison of immunostaining and DNA sequence alterations showed a significant association between overexpression and mutations (P = 0. 0037) in cases of CIS without invasion, and similarly between overexpression and mutations in cases of CIS with invasion (P = 0. 007). p53 mutations and p53 overexpression were relatively common in intraductal breast carcinomas but were not observed in adjacent normal breast lobules or ducts in 9 cases available for DNA analysis. The frequency of p53 alterations when comparing breast CIS with and without an invasive component indicated that p53 mutations usually occur before invasion during the progression of breast cancer, as is observed for a number of other adult solid tumors.

Base Sequence↗
PubMed2000

Bacteriology and antibiotic therapy in congenital nasolacrimal duct obstruction.

AIMS: To determine the current bacteriology of mucopurulent discharge in congenital nasolacrimal duct obstruction (CNDO), the in vitro response to different antibiotics and clinical effectiveness of the antibiotics used to relieve babies from mucopurulent discharge. METHODS: A clinical study evaluated the effectiveness of local antibiotic agents clinically and in vitro. 50 samples were obtained from the lacrimal sac in 47 young children with CNDO. The patients' mean age was 21.45 +/- 17.09 months. The cultures were incubated and the infectious agents isolated. Sensitivity testing was performed in each case, testing 10 different local antibiotics. A control group of 10 babies expected for cataract surgery was constituted. RESULTS: Cultures were positive for bacteria from 72.64% of the samples. 73 isolates were recovered from the 50 samples. The bacterial species most frequently cultured was Streptococcus pneumoniae, representing 35.4% of the isolates, followed by Haemophilus influencae (19.6%). The sensitivity testing revealed ofloxacin and tetracycline to be the most effective drugs as monotherapy. Clinically the combination of bacitracin and neomycin, primarily used in half of the patients as initial therapy, was successful in curing the dacryocystitis in 82.5% of all patients. CONCLUSION: Chronic dacryocystitis due to CNDO is associated with an equal proportion of Gram positive and negative bacteria, which can be treated with a high effectiveness by a combination drug of bacitracin and neomycin.

Anti-Bacterial Agents↗
PubMed2000

Monoclonal antibody probes for p21WAF1/CIP1 and the INK4 family of cyclin-dependent kinase inhibitors.

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle control. Although not fully understood at present, the functions of the individual members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, responses to genotoxic stress, regulation of cellular differentiation, and senescence. In addition, the seven currently known CKIs qualify as either established or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, we report the isolation and characterization of a panel of 10 mouse monoclonal antibodies (MAbs) that specifically recognize p21WAF1/CIP1 (p21) or the individual members of the INK4 family of CKIs: p15INK4b (p15), p16INK4a (p16), p18INK4c (p18), or p19INK4d (p19). These antibodies are proving to be invaluable molecular probes for analyses of protein abundance, subcellular localization, interacting cellular proteins, and ultimately the function(s) of these cell cycle regulators. Epitopes targeted by the antibodies were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. Individual MAbs of our series recognize distinct pools of the respective CKIs, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Together, these antibodies represent useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivation of these (candidate) tumor suppressors in human malignancies.

Animals↗
PubMed2000

Involvement of Myc activity in a G(1)/S-promoting mechanism parallel to the pRb/E2F pathway.

The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRbDeltacdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G(1)/S delay caused by pRbDeltacdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRbDeltacdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G(1)/S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G(1)/S-promoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.

CDC2-CDC28 Kinases↗
PubMed2000

Nonperiodic activity of the human anaphase-promoting complex-Cdh1 ubiquitin ligase results in continuous DNA synthesis uncoupled from mitosis.

Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae and Drosophila spp., triggers exit from mitosis and during G(1) prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G(1)/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G(1)/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27(Kip1) cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G(1)/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

Anaphase-Promoting Complex-Cyclosome↗
PubMed1999

Accumulation of cyclin B1 requires E2F and cyclin-A-dependent rearrangement of the anaphase-promoting complex.

In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Here, using an in vivo assay to measure protein stability in real time during the cell cycle, we show that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A resulted in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.

Anaphase↗
PubMed1999

p16INK4a, but not constitutively active pRb, can impose a sustained G1 arrest: molecular mechanisms and implications for oncogenesis.

p16ink4 and pRb, two components of a key G1/S regulatory pathway, and tumor suppressors commonly targeted in oncogenesis, are among the candidates for gene therapy of cancer. Wild-type p16 and a constitutively active pRb(delta cdk) mutant both blocked G1 in short-term experiments, but only p16 imposed a sustained G1 arrest. Unexpectedly, cells conditionally exposed to pRb(delta cdk) entered S phase after 2 days, followed by endoreduplication between days 4-6. The distinct phenotypes evoked by p16 vs pRb(delta cdk) appear mediated by cyclin E/CDK2 which, while active in the pRb(delta cdk)-expressing cells, became rapidly inhibited through restructuring diverse cyclin/CDK/p21 complexes by p16. These results provide novel insights into the roles of p16, pRb and cyclin E in G1/S control and multistep oncogenesis, with implications for gene therapy strategies.

Animals↗
PubMed1999

Phosphorylation of mammalian CDC6 by cyclin A/CDK2 regulates its subcellular localization.

Cyclin-dependent kinases (CDKs) are essential for regulating key transitions in the cell cycle, including initiation of DNA replication, mitosis and prevention of re-replication. Here we demonstrate that mammalian CDC6, an essential regulator of initiation of DNA replication, is phosphorylated by CDKs. CDC6 interacts specifically with the active Cyclin A/CDK2 complex in vitro and in vivo, but not with Cyclin E or Cyclin B kinase complexes. The cyclin binding domain of CDC6 was mapped to an N-terminal Cy-motif that is similar to the cyclin binding regions in p21(WAF1/SDI1) and E2F-1. The in vivo phosphorylation of CDC6 was dependent on three N-terminal CDK consensus sites, and the phosphorylation of these sites was shown to regulate the subcellular localization of CDC6. Consistent with this notion, we found that the subcellular localization of CDC6 is cell cycle regulated. In G1, CDC6 is nuclear and it relocalizes to the cytoplasm when Cyclin A/CDK2 is activated. In agreement with CDC6 phosphorylation being specifically mediated by Cyclin A/CDK2, we show that ectopic expression of Cyclin A, but not of Cyclin E, leads to rapid relocalization of CDC6 from the nucleus to the cytoplasm. Based on our data we suggest that the phosphorylation of CDC6 by Cyclin A/CDK2 is a negative regulatory event that could be implicated in preventing re-replication during S phase and G2.

Animals↗
PubMed1999

Perspective: defects in cell cycle control and cancer.

The past several years have witnessed a dramatic accumulation of experimental and clinical evidence supporting the notion that the cell cycle machinery is commonly targeted on oncogenesis. While numerous cell cycle regulators qualify as proto-oncogenes or tumour suppressors and their aberrations may provide direct proliferative advantage to cancer cells, defects in checkpoint mechanisms act more indirectly yet affect both tumour progression and response to anticancer therapy. In this review, the ways that cell cycle defects contribute to oncogenesis are briefly illustrated and the emerging benefits of the newly gained insights into the cell cycle clock for clinical oncology are critically considered. Given the many reviews on the subject, emphasis is put on concepts rather than comprehensive treatment of the selected topics, with particular attention given to controversial issues, unorthodox phenomena, and the challenge facing the 'cell cycle and cancer field' at the transition to the next millennium.

Cell Cycle↗
PubMed1999

Dissecting functions of the retinoblastoma tumor suppressor and the related pocket proteins by integrating genetic, cell biology, and electrophoretic techniques.

The members of the 'pocket protein' family, comprising the retinoblastoma tumor suppressor (pRB) and its relatives, p107 and p130, negatively regulate cell proliferation and modulate fundamental biological processes including embryonic development, differentiation, homeostatic tissue renewal, and defense against cancer. The large, multidomain pocket proteins act by binding a plethora of cell fate-determining and growth-stimulatory proteins, the most prominent of which are the E2F/DP transcription factors. These protein-protein interactions are in turn regulated by carefully orchestrated phosphorylation events on multiple serine and threonine residues of pRB, p107, and p130, events which are carried out, at least in part, by the cyclin-dependent kinases that form the key elements of the cell cycle machinery. Here we discuss the recently obtained new insights into the diverse functions of the pRB family, and show examples of how integration of genetic, cell biology, and a range of electrophoretic approaches help to advance our understanding of the biological roles played by the pocket proteins in both normal and cancer cells.

Amino Acid Sequence↗
PubMed1999

Centrosome duplication in mammalian somatic cells requires E2F and Cdk2-cyclin A.

Centrosome duplication is a key requirement for bipolar spindle formation and correct segregation of chromosomes during cell division. In a manner highly reminiscent of DNA replication, the centrosome must be duplicated once, and only once, in each cell cycle. How centrosome duplication is regulated and coordinated with other cell-cycle functions remains poorly understood. Here, we have established a centrosome duplication assay using mammalian somatic cells. We show that centrosome duplication requires the activation of E2F transcription factors and Cdk2-cyclin A activity.

Animals↗